The overall goal of this project is to investigate the signaling pathways, particularly ion signaling pathways, that regulate apoptosis. We studied cells that respond differently to apoptotic induction following growth factor removal (i.e. low serum). Early stage preneoplastic, immortal cells (sup+), show a high susceptibility to induction of apoptosis, whereas late stage preneoplastic cells (sup-) are relatively resistant to apoptosis following serum reduction to 0.2%. We investigated whether altered Ca2+ homeostasis is causally involved in apoptosis. We showed that a sustained increase in cytosolic free Ca2+ (Cai) does not precede apoptosis. Differences in endoplasmic reticulum (ER) calcium between sup+ cells and sup- were determined by measuring thapsigargin releasable Ca2+ in the presence of 10% and 0.2% serum. Sup+ cells in low serum exhibit decreased endoplasmic reticulum calcium levels and subsequent DNA laddering, indicative of apoptosis. We are investigating signals that are downstream of decreased ER calcium (ceramide and NF-kB). Studies show that following 4 hours of low serum treatment, a time when both decreased calcium entry and decreased ER calcium are observed, no change in ceramide is detected in either sup+ or sup- cells. At 16 hours after serum reduction the sup+ cells, which undergo apoptosis, exhibited a 60-70% increase in ceramide, while the sup- cells, which do not undergo apoptosis, showed no change in ceramide levels. These data suggest that changes in ceramide are later and potentially downstream of changes in ER calcium. It is important to show whether changes in ceramide are important in causing apoptosis or whether they are not just a result of apoptosis. Addition of ceramide to sup- cells in low serum resulted in apoptosis, consistent with a role for ceramide in low serum induced apoptosis. Interestingly, addition of ceramide to sup+ cells in 10% serum resulted in a G2/M arrest. Thus addition of ceramide is not sufficient to induce apoptosis, but seems to require an additional signal which is stimulated by low serum. To substantiate that ceramide lies on the same signaling cascade as decreased ER calcium, it was necessary to show a causal relationship between depletion of ER calcium and elevation of ceramide. Depletion of ER calcium with thapsigargin also caused an increase in ceramide in both sup+ and sup- cells. Furthermore if we blocked the decrease in ER calcium in low serum, by raising extracellular calcium we blocked the rise in ceramide. Also there are reports that a decrease in ER calcium can lead to activation of NF-kB. We therefore measured NF-kB, but we found that sup+ cells showed little activation of NF-kB in either 10% or 0.2% serum. Thus, in spite of the decreased ER Ca2+ in sup+ cells in low serum we did not observe an elevation of NF-kB. In contrast, sup- cells showed considerable NF-kB activation in 10% and 0.2% serum (~5-10x higher than sup+). This increase in activated NF-kB in sup- cells is not altered by addition of proteosome inhibitors. The increase in basal NF-kB in sup- compared to sup+ cells could be related to the increase in basal Cai in sup- cells, consistent with reports that calcium can enhance degradation of I-kB and thus activate NF-kB. To test this hypothesis we lowered basal calcium in sup- cells in 10% serum by addition of the intracellular calcium chelator BAPTA-AM. We confirmed using fura-2 and fluorescent microscopy that BAPTA lowered basal calcium by 31%; this reduction in calcium was accompanied by a 65% reduction in NF-kB binding activity and a reduction in IkB kinase. In sup- cells in low serum, concomitant with the lowering of cytosolic Ca2+ and NF-kB binding activity, we found a significant increase in caspase 3 activity. Furthermore, in sup+ cells, raising extracellular Ca2+ resulted in activation of IkB kinase, enhanced NF-kB binding activity and reduced apoptosis. These data suggest that an increase in cytosolic Ca2+ leads to an activation of NF-kB and resistance to apoptosis.